Construction of LEU1 gene deleted Saccharomyces cerevisiae based on CRISPR-Cas9 system for brewing low degree of drunkenness rice wine
In order to explore the effect of the deletion of LEU1 gene of isopropyl malate synthase in Saccharomyces cerevisiae on the production of higher alcohols in rice wine brewing,the recombinant plasmids p414-Cas9-BleoR and p426-gRNA.LEU1-kanMX were constructed and electrotrans-formed into S.cerevisiae XF0,and the LEU1 gene deletion S.cerevisiae XF0-L was constructed by clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9(CRISPR-Cas9)system.The original strain XF0 and recombinant strain XF0-L1 were used for medium simulation fermentation and rice wine fermentation,and the physicochemical indexes and higher alcohols of fermented wine were determined.The results showed that there was no significant difference in the physicochemical indexes between strain XF0-L and XF0 simulated fermented wine(P＞0.05),indicating that the deletion of LEU1 gene had no effect on the fermentation performance of S.cerevisiae.Compared with the original strain XF0,the content of n-propanol(26.27 mg/L)and isobutanol(88.64 mg/L)in the simulated fermented wine of recombinant strain XF0-L increased by 21.96％and 85.25％,respectively,and the content of isoamyl alcohol(141.82 mg/L),isoamyl alcohol and n-propanol ratio(5.39)and isoamyl alcohol and isobutanol ratio(1.60)decreased by 15.53％,30.99％and 54.42％,respectively.Compared with the original strain XF0,the content of n-propanol(268.67 mg/L)and isobutanol(992.33 mg/L)in the fermented rice wine of recombinant strain XF0-L increased by 14.17％and 52.90％,respectively,while the content of isoamyl alcohol(1 016.33 mg/L),isoamyl alcohol and n-propanol ratio(3.78)and isoamyl alcohol and isobutanol ratio(1.02)decreased by 13.58％,24.40％and 43.65％,respectively.The results indicated that the recombinant strain XF0-L could significantly reduce isoamyl alcohol and n-propanol ratio,isoamyl alcohol and isobutanol ratio to reduce the drunkenness(P＜0.05).
Saccharomyces cerevisiaeCRISPR-Cas9LEU1 genehigher alcoholslow degree of drunkenness rice wine
万方数据
维普
